Novel Auger-Electron-Emitting 191Pt-Labeled Pyrrole-Imidazole Polyamide Targeting MYCN Increases Cytotoxicity and Cytosolic dsDNA Granules in MYCN-Amplified Neuroblastoma.

Auger electrons can cause nanoscale physiochemical damage to specific DNA sites that play a key role in cancer cell survival. Radio-Pt is a promising Auger-electron source for damaging DNA efficiently because of its ability to bind to DNA. Considering that the cancer genome is maintained under abnormal gene amplification and expression, here, we developed a novel 191Pt-labeled agent based on pyrrole-imidazole polyamide (PIP), targeting the oncogene MYCN amplified in human neuroblastoma, and investigated its targeting ability and damaging effects. A conjugate of MYCN-targeting PIP and Cys-(Arg)3-coumarin was labeled with 191Pt via Cys (191Pt-MYCN-PIP) with a radiochemical purity of >99%. The binding potential of 191Pt-MYCN-PIP was evaluated via the gel electrophoretic mobility shift assay, suggesting that the radioagent bound to the DNA including the target sequence of the MYCN gene. In vitro assays using human neuroblastoma cells showed that 191Pt-MYCN-PIP bound to DNA efficiently and caused DNA damage, decreasing MYCN gene expression and MYCN signals in in situ hybridization analysis, as well as cell viability, especially in MYCN-amplified Kelly cells. 191Pt-MYCN-PIP also induced a substantial increase in cytosolic dsDNA granules and generated proinflammatory cytokines, IFN-α/ß, in Kelly cells. Tumor uptake of intravenously injected 191Pt-MYCN-PIP was low and its delivery to tumors should be improved for therapeutic application. The present results provided a potential strategy, targeting the key oncogenes for cancer survival for Auger electron therapy.

DNA Repair Inhibitors: Potential Targets and Partners for Targeted Radionuclide Therapy.

The present review aims to explore the potential targets/partners for future targeted radionuclide therapy (TRT) strategies, wherein cancer cells often are not killed effectively, despite receiving a high average tumor radiation dose. Here, we shall discuss the key factors in the cancer genome, especially those related to DNA damage response/repair and maintenance systems for escaping cell death in cancer cells. To overcome the current limitations of TRT effectiveness due to radiation/drug-tolerant cells and tumor heterogeneity, and to make TRT more effective, we propose that a promising strategy would be to target the DNA maintenance factors that are crucial for cancer survival. Considering their cancer-specific DNA damage response/repair ability and dysregulated transcription/epigenetic system, key factors such as PARP, ATM/ATR, amplified/overexpressed transcription factors, and DNA methyltransferases have the potential to be molecular targets for Auger electron therapy; moreover, their inhibition by non-radioactive molecules could be a partnering component for enhancing the therapeutic response of TRT.

Dynamic imaging analysis reveals Auger electron-emitting radio-cisplatin induces DNA damage depending on the cell cycle.

Auger electrons can induce nanoscale physiochemical damage to DNA. The present study reports a sequential and systematic evaluation of the relationship between DNA damage such as double-strand breaks (DSBs) and the cell cycle for the Auger electron-emitting agent radiolabeled cisplatin with DNA binding ability. For dynamic imaging analysis, we used U2OS-derived cancer cells expressing two fluorescent fusion proteins: tumor-suppressor p53 binding protein 1 with a green fluorescent protein (53BP1-EGFP) and proliferating cell nuclear antigen with a red fluorescent protein (PCNA-DsRed). Time-lapse images of the cells were quantitatively analyzed using the ImageJ software with the deepImageJ plugin and the Google Colaboratory platform. From the middle-to-late G1 phase, around the G1-to-S phase transition, we found increased 53BP1 foci in cells treated with the radio-cisplatin. The radio-cisplatin caused significantly more DSBs than the nonradioactive cisplatin and saline in the G1 phase but not in the other phases. These results indicate that Auger electron-induced DNA damage, including DSBs, depends on the cell cycle. The G1 phase, which is associated with low DNA repair capacity and high radiosensitivity, is a promising target; thus, combining radiolabeled cisplatin with agents that arrest cells in the G1 phase could improve the DNA-damaging effect of Auger electrons and their therapeutic efficacy.

Precise quantitative evaluation of pharmacokinetics of cisplatin using a radio-platinum tracer in tumor-bearing mice.

OBJECTIVE: The platinum-based antineoplastic drug cisplatin is commonly used for chemotherapy in clinics. This work aims to demonstrate a radio-platinum tracer is useful for precisely quantifying small amounts of platinum in pharmacokinetics studies. METHODS: A cisplatin radiotracer (radio-cisplatin) was synthesized, and a comprehensive evaluation of cisplatin over 7 days after its intravenous injection into nude mice bearing a subcutaneous lung tumor (H460) was conducted. RESULTS: A biphasic retention curve in the whole body and blood was observed [ T1/2 (α) = 1.14 h, T1/2 (ß) = 5.33 days for the whole body, and T1/2 (α) = 23.9 min, T1/2 (ß) = 4.72 days for blood]. The blood concentration decreased within 1 day after injection. Most of the intact cisplatin was excreted via the kidneys in the early time points, and a small part was distributed in tissues including tumors. The plasma protein binding rate of cisplatin increased rapidly after injection, and the protein-bound cisplatin remained in the blood longer than intact cisplatin. The peak uptake in H460 tumors was 4.7% injected dose per gram at 15 min after injection, and the area under the curve (AUC 0-7 days ) was approximately one-half to one-third of the AUC 0-7 days in the kidneys, liver, and bone, where some toxicity is observed in humans. CONCLUSION: The radio-platinum tracer revealed the highly quantitative biodistribution of cisplatin, providing insights into the properties of cisplatin, including its adverse effects. The tracer enables a precise evaluation of pharmacokinetics for platinum-based drugs with high sensitivity.

Development of Novel 191Pt-Labeled Hoechst33258: 191Pt Is More Suitable than 111In for Targeting DNA.

This study aims to establish new labeling methods for no-carrier-added radio-Pt (191Pt) and to evaluate the in vitro properties of 191Pt-labeled agents compared with those of agents labeled with the common emitter 111In. 191Pt was complexed with the DNA-targeting dye Hoechst33258 via diethylenetriaminepentaacetic acid (DTPA) or the sulfur-containing amino acid cysteine (Cys). The intranuclear fractions of 191Pt- and 111In-labeled Hoechst33258 were comparable, indicating that the labeling for 191Pt via DTPA or Cys and the labeling for 111In via DTPA worked equally well. 191Pt showed a DNA-binding/cellular uptake ratio of more than 1 order of magnitude greater than that of 111In. [191Pt]Pt-Hoechst33258 labeled via Cys showed a higher cellular uptake than that labeled via DTPA, resulting in a very high DNA-binding fraction of [191Pt]Pt-Cys-Hoechst33258 and extensive DNA damage. Our labeling methods of radio-Pt, especially via Cys, promote the development of radio-Pt-based agents for use in Auger electron therapy targeting DNA.

Development of a Stable Peptide-Based PET Tracer for Detecting CD133-Expressing Cancer Cells.

CD133 has been recognized as a prominent biomarker for cancer stem cells (CSCs), which promote tumor relapse and metastasis. Here, we developed a clinically relevant, stable, and peptide-based positron emission tomography (PET) tracer, [64Cu]CM-2, for mapping CD133 protein in several kinds of cancers. Through the incorporation of a 6-aminohexanoic acid (Ahx) into the N terminus of a CM peptide, we constructed a stable peptide tracer [64Cu]CM-2, which exhibited specific binding to CD133-positive CSCs in multiple preclinical tumor models. Both PET imaging and ex vivo biodistribution verified the superb performance of [64Cu]CM-2. Furthermore, the matched physical and biological half-life of [64Cu]CM-2 makes it a state-of-the-art PET tracer for CD133. Therefore, [64Cu]CM-2 PET may not only enable the longitudinal tracking of CD133 dynamics in the cancer stem cell niche but also provide a powerful and noninvasive imaging tool to track down CSCs in refractory cancers.

Transition-metal-free nucleophilic 211At-astatination of spirocyclic aryliodonium ylides.

The transition-metal-free 211At-astatination of spirocyclic aryliodonium ylides via a nucleophilic aromatic substitution reaction is described. This method enables the preparation of 211At-radiolabeled compounds derived from multi-functionalized molecules and heteroarenes in good to excellent radiochemical yields.

Synthesis of [211At]4-astato-L-phenylalanine by dihydroxyboryl-astatine substitution reaction in aqueous solution.

Astatine-211 (211At)-labeled phenylalanine is expected to be a promising agent for targeted alpha-particle therapy for the treatment of patients with glioma. The existing reactions to prepare the labeled compound usually require organic solvents and metals that are toxic and hazardous to the environment. In this study, we developed a novel method wherein astatination was realized via the substitution of 211At for a dihydroxyboryl group coupled to phenylalanine. [211At]4-astato-L-phenylalanine was obtained as the carrier-free product in aqueous medium in high radiochemical yields (98.1 ± 1.9%, n = 5). The crude reaction mixture was purified by solid-phase extraction, and the radiochemical purity of the product was 99.3 ± 0.7% (n = 5). The high yield and purity were attributed to the formation of [211At]AtI and AtI2- as the reactive intermediates in the astatination reaction. The reaction did not require any organic solvents or toxic reagents, suggesting that this method is suitable for clinical applications.

Synthesis of no-carrier-added [188, 189, 191Pt]cisplatin from a cyclotron produced 188, 189, 191PtCl42- complex.

We developed a novel method for production of no-carrier-added (n.c.a.) [188, 189, 191Pt]PtIICl42- from an Ir target material, and then synthesized n.c.a. [*Pt]cis-[PtIICl2(NH3)2] ([*Pt]cisplatin) from [*Pt]PtIICl42-. [*Pt]PtIICl42- was prepared as a synthetic precursor of n.c.a. *Pt complex by a combination of resin extraction and anion-exchange chromatography after the selective reduction of IrIVCl62- with ascorbic acid. The ligand-substitution reaction of Cl with NH3 was promoted by treating n.c.a. [*Pt]PtIICl42- with excess NH3 and heating the reaction mixture, and n.c.a. [*Pt]cisplatin was successfully produced without employing precipitation routes. After this treatment, [*Pt]cisplatin was isolated through preparative HPLC with a radiochemical purity of 99 + % at the end of synthesis (EOS).

In Vitro Evaluation of No-Carrier-Added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons.

Due to their short-range (2-500 nm), Auger electrons (Auger e-) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Auger e-, it remains challenging to maximize the interaction between Auger e- and DNA. To assess the DNA-damaging effect of Auger e- released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by immunofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incubation), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by the release of Auger e- very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e-.

Production of 191Pt from an iridium target by vertical beam irradiation and simultaneous alkali fusion.

We have developed a new method for producing 191Pt from an iridium target. Alkali fusion of iridium was successfully performed using a vertical beam irradiation method and a mixed target of Ir and Na2O2, which resulted in easy dissolution of the irradiated iridium target. A trace amount of PtⅣCl62- was isolated from bulk IrⅣCl62- by solvent extraction and anion exchange chromatography. The production yield of 191Pt was 7.1 ±â€¯0.4 (MBq/µA h, EOB) by proton irradiation at 30 MeV. The radioplatinum product (n.c.a.) was prepared at a radiochemical purity of 97% for PtⅣCl62-, and 95% for PtⅡCl42-, respectively.

Excitation functions of proton- and deuteron-induced nuclear reactions on natural iridium for the production of 191Pt.

We studied the excitation functions of residual radionuclides produced via proton and deuteron bombardment on natural iridium in the energy ranges of 30-15 MeV and 50-15 MeV, respectively. A conventional stacked-foil activation technique combined with HPGe γ-ray spectrometry was used to measure the excitation functions for 189, 191Pt and 189, 190g, 192g, 194gIr radionuclide production. Theoretical thick target yields were estimated to be 172 MBq/µA h and 192 MBq/µA h via the 193Ir(p,3n)191Pt reaction at 29.6-17.5 MeV and the 193Ir(d,4n)191Pt reaction at 40.3-23.8 MeV, respectively. The feasibility of 191Pt production from an iridium target was discussed, and compared with previously reported methods for the production of 191Pt.